Short-Wave Enhances Mesenchymal Stem Cell Recruitment in Fracture Healing by Increasing HIF-1 in Callus

Ye et al. Stem Cell Research & Therapy (2020) 11:382 https://doi.org/10.1186/s13287-020-01888-0

RESEARCH Open Access Short-wave enhances mesenchymal stem cell recruitment in fracture healing by increasing HIF-1 in callus Dongmei Ye1*† , Chen Chen2†, Qiwen Wang1,3, Qi Zhang1, Sha Li1 and Hongwei Liu1

Abstract Background: As a type of high-frequency electrotherapy, a short-wave can promote the fracture healing process; yet, its underlying therapeutic mechanisms remain unclear. Purpose: To observe the effect of Short-Wave therapy on mesenchymal stem cell (MSC) homing and relative mechanisms associated with fracture healing. Materials and methods: For in vivo study, the effect of Short-Wave therapy to fracture healing was examined in a stabilized femur fracture model of 40 SD rats. Radiography was used to analyze the morphology and microarchitecture of the callus. Additionally, fluorescence assays were used to analyze the GFP-labeled MSC homing after treatment in 20 nude mice with a femoral fracture. For in vitro study, osteoblast from newborn rats simulated fracture site was first irradiated by the Short-Wave; siRNA targeting HIF-1 was used to investigate the role of HIF-1. Osteoblast culture medium was then collected as chemotaxis content of MSC, and the migration of MSC from rats was evaluated using wound healing assay and trans-well chamber test. The expression of HIF-1 and its related factors were quantified by q RT-PCR, ELISA, and Western blot. Results: Our in vivo experiment indicated that Short-Wave therapy could promote MSC migration, increase local and serum HIF-1 and SDF-1 levels, induce changes in callus formation, and improve callus microarchitecture and mechanical properties, thus speeding up the healing process of the fracture site. Moreover, the in vitro results further indicated that Short-Wave therapy upregulated HIF-1 and SDF-1 expression in osteoblast and its cultured medium, as well as the expression of CXCR-4, β-catenin, F-actin, and phosphorylation levels of FAK in MSC. On the other hand, the inhibition of HIF-1α was significantly restrained by the inhibition of HIF-1α in osteoblast, and it partially inhibited the migration of MSC. Conclusions: These results suggested that Short-Wave therapy could increase HIF-1 in callus, which is one of the crucial mechanisms of chemotaxis MSC homing in fracture healing. Keywords: Mesenchymal stem cells, Short-Wave therapy, Recruitment, Fracture, HIF-1

* Correspondence: [email protected] †Dongmei Ye and Chen Chen contributed equally to this work. 1Department of Rehabilitation, Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, China Full list of author information is available at the end of the article

© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 2 of 16

Introduction fracture in the animal model and assessed the healing of Fracture healing is a biologically optimized process. fracture using the radiographs. Also, in vivo biolumines- Mesenchymal stem cells (MSCs) are multipotent stromal cent assays and callus histo-immunofluorescence were cells that can differentiate into multiple cell types such applied to evaluate the MSC migration. HIF-1 and re- as chondrocytes and osteocytes and thus have an essen- lated factors were also detected. siRNA targeted HIF-1α tial role in the bone healing process [1]. While fracture was transfected to clarify its effect in Short-Wave ther- healing, circulating MSC can receive signals from the in- apy (Fig. 1). This study aimed to investigate the impact jured tissue and migrate to the damaged sites. Over the of Short-Wave therapy on MSC recruitment and explore last decade, several strategies for fracture healing have the underlying mechanisms of Short-Wave therapy on been investigated, including the stimulation of endogen- fracture healing. ous stem cell populations from the mature body [2, 3]. An alternative strategy is the use of exogenous stem Materials and methods cells, which can be obtained from connective tissues and MSC isolation and identification are controlled by the expression of molecules during MSC was isolated from femurs of 3-week-old male SD MSC expansion, such as CXCR4 and complement 1q rats, as previously described [22]. Before performing any (C1q) [4, 5], or by certain chemicals [6], such as valpro- experiment, cells were passaged for 3 to 6 times. The ate or lithium, which are involved in MSC homing and surface marker expressions of MSC, including CD90, can trigger the expression of certain key factors. Since CD44, CD34, CD45, and CD11b/c, were analyzed by the mobilization is a kind of directional migration, both flow cytometry assay, as described in the previous study endogenous and exogenous MSC recruitment is related [23, 24]. to the condition of the fracture site. Currently, re- searchers are focusing on improving the condition of Animal grouping MSC recruitment by using biological agents. Clinical- A total of 40 male SD rats, 8–12 weeks old, weighing standard platelet products loaded membranes [7] and 350–500 g, were obtained from Laboratory Animal Cen- naringin [8] successfully support MSC colony formation ter of Dalian Medical University, China. Rats were and promote MSC migration in vitro. Furthermore, housed in an environment with a temperature of 22 ± MSC migration can be improved under hypoxic condi- 1 °C, a relative humidity of 50 ± 1%, and a light/dark tions. Previous studies have suggested that hypoxic con- cycle of 12/12 h. ditions decrease the MMP secretion and increase After 1 week of adaptation, 40 SD rats were accurately CXCR4 expression [9, 10]. However, the effect of phys- weighed and randomly divided into two groups (n = 20/ ical agency therapy on MSC migration has not received group): Short-Wave treatment group (SW) and control adequate attention. group (Con). All rats underwent surgery to establish the Short-Wave therapy is a type of high-frequency elec- femoral shaft fracture and intramedullary fixation model. trotherapy, which can promote the fracture healing For in vivo bioluminescent assays to the MSC homing, process [11, 12]. At high frequencies, the electromag- 20 nude mice of femoral shaft fracture were injected MSC netic energy is converted to thermal energy, which can labeled by the GFP (MG) and then randomly divided into induce heat (temperature over 40 °C) to the treated area two groups, including a Short-Wave treatment group of the body [13], where the heating process affects blood (MG+SW) and a control group (MG) (n = 10/group). flow [14] and decreases pain [15]. Accumulating evidence has indicated that both hypoxia and hypoxia- Stabilized fracture model driven angiogenesis can be regulated by thermal therapy Stabilized right femur fractures were established by [16]. The hypoxia-inducible factor (HIF) involved signal- intramedullary fixation in 8–12-week-old male SD rats. ing pathway is activated under hypoxia. HIF protein, es- A 0.25-mm titanium alloy pin was inserted inside the pecially HIF-1α, has been associated with MSC medullar canal of the femur. A three-point bending de- migration and differentiation [17]. As a critical transcrip- vice was then used to produce closed fractures of the tional regulator, HIF-1 can regulate the expression of femoral shaft with a standardized force [25]. In 20 male multiple cytokines, such as stromal cell-derived factor 1 nude mice, transverse osteotomy with a 1-mm bone (SDF-1) [10, 18] and focal adhesion kinase (FAK) [19], fracture was created in the middle of the right femur in which has a central role in the adaptation of MSC to the same way. Buprenorphine was subcutaneously ad- hypoxia [20, 21]. ministered (0.5 mg/kg) for pain control. The present study explored the effects of Short-Wave therapy on HIF-1 expression in fracture and MSC re- Cell injection cruitment from peripheral blood during fracture healing. All the 20 nude mice received injections with 5 × 106/ Accordingly, we applied Short-Wave treatment on the 50 μl GFP-labeled MSC (MG) (C57BL/6 mouse strain) Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 3 of 16

Fig. 1 Flowchart of the study design. Stabilized femur fractures were established in 40 SD rats. Radiographs and micro-CT analysis examined the effect of Short-Wave on fracture healing. The expression of HIF-1 and other factors in callus was tested by q RT-PCR. SDF-1 in plasma was evaluated by ELISA. To analyze the MSC migration in healing, in vivo fluorescence assays and immunofluorescence were used after treatment in

20 nude mice with a femoral fracture. For in vitro study, osteoblast simulated fracture site was first irradiated by the Short-Wave; CoCl2 in medium stimulated hypoxia condition; siRNA targeting HIF-1 was used to investigate the role of HIF-1. Osteoblast culture medium was then collected as chemotaxis content of MSC, and the migration of MSC was evaluated using wound healing assay and trans-well chamber test. The expression of HIF-1 and its related factors were quantified by q RT-PCR, ELISA, and Western blot. SW Short-Wave treatment, MG GFP-labeled MSC. The image element in the flowchart mainly comes from https://app.biorender.com/

(CYAGEN Company, China) 3 days after the femoral turn around. The treatment regimen was applied to the fracture. Cells were injected by tail vein using a right thigh. The Short-Wave generator operated at a fre- microinjector. quency of 27.12 MHz. A micro-heat continuous-wave Short-Wave exposure for 10 min was applied once a day. Short-Wave treatment A similar procedure was carried out in the control Three days after the operation, Short-Wave therapy was group; however, the device was turned off. applied in the Short-Wave treatment group. Briefly, ani- For the in vitro study, the isolated rat osteoblasts in mals were fixed in an apparatus so that they could not the treatment group underwent Short-Wave irradiation. Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 4 of 16

The protocol of Short-Wave therapy irradiation on cells positive cells were automatically counted in five fields on was based on a previous research [26]. The two non- × 100 magnification by ImageJ software. contact applicators were of perpendicular contraposition and 10 cm away from each other. The cell culture flask was Enzyme-linked immunosorbent assay (ELISA) placed in the middle of the applicators. A micro-heat The plasma of SD rats received by heart puncture at continuous-wave Short-Wave exposure for 90 min was ap- days 3, 7, and 14 after the operation (n = 4/group/time plied twice on day 1 in the open air at 37 °C. The control point). The concentration SDF-1 in plasma of SD rats group received a sham Short-Wave treatment by turning was analyzed using citrulline ELISA kit (CSB-E13414r, off the Short-Wave generator. On day 2, the culture Cusabio Biotech, China). In culture media of osteoblasts medium from each group was collected for HIF-1 and (SD rat strain), the concentration of HIF-1 (SEA798Ra, SDF-1 protein-level analysis, and the medium was used in USCN, China) and SDF-1 (SEA122Ra, USCN, China) MSC cell wound healing assay and trans-well chamber test. content was also detected using the ELISA kit.

Radiographic assessment Quantitative reverse transcription-polymerase chain The fracture healing was assessed by plain anteroposter- reaction (q RT-PCR) ior radiographs at days 7, 14, 21, and 28. The same X- Callus of sacrificed SD rats was collected and snap- ray machine and settings were used for all radiographs frozen in liquid nitrogen at days 3, 7, and 14 after the every 7 days after fracture. Bone defects were analyzed operation (n = 4/group/time point). RNA isolation and using X-ray software. subsequent cDNA synthesis (Bio-Rad, 170-8891) were Micro-CT scanning was performed to assess callus. performed as previously described [31]. A total of 50 ng Quantification for the volumes of the bony calluses as of cDNA was amplified with custom-designed q RT- determined as previously described [27, 28]. The region PCR primers (Table 1). A melt curve was generated to of interest was set within 800 μm (50 slices) around the analyze the purity of amplification products. The expres- defect edge. We applied a fixed threshold of less than sion levels of mRNA were normalized to the average of 330 for new calcified cartilage or unmineralized cartil- β-actin. Relative expression of mRNA was evaluated by age. Three-D microstructural image data were recon- using the comparative CT method (ΔΔCt) [32]. structed by Inveon Research Workplace software. After 3D reconstruction, bone volume fraction (BV/TV) was automatically determined to confirm fracture healing. Osteoblast culture and identification Osteoblasts were obtained from calvaria of 1-day-old In vivo fluorescence assays neonatal SD rats using the method of collagenase- Ten nude mice per group received anesthesia and were pancreatic enzyme digestion as detailed in reference examined by the IVIS imaging system at day 7 after the [33]. After two passages, alkaline phosphatase staining surgery. The identical parameter settings were used for was utilized to identify the osteoblast cells. all samples: f number 1, field of view 22, binning factor 18, and luminescent exposure (seconds) 10. The IVIS Small interference RNA transfection imaging examination and rates of photons were calcu- We inhibited HIF-1α expression by siRNA in osteoblasts lated and performed according to methods reported in a (SD rat strain). Synthetic siRNA oligonucleotide specific previous study [29]. for HIF-1α (NM_024359) (5′ to 3′: UUUAUCAAGA UGGGAGCUCTT) and nontargeting siRNA were ob- Histological analysis and immunofluorescence imaging tained from Sangon (Shanghai, China). Twenty nude mice (MG: n =10,MG+SW: n = 10) were sacrificed for histological analysis and immunofluores- Table 1 Oligonucleotide product size and accession numbers cence imaging at day 28 after the operation. The femoral for q RT-PCR bone of nude mice and rats were sectioned, preserved, Gene Product size Accession number decalcified, and embedded in paraffin along the longitu- HIF-1 105 NM_021704 dinal axis. For morphological analysis, 5-μmsliceswere sectioned, deparaffinized, and stained using hematoxylin SFD-1 126 NM_001033 and eosin. The immunofluorescence staining was per- FAK 130 NM_013081 formed as previously described [30]. Tissue slides of nude F-actin 127 NM_001109 mice were stained with antibodies (A0516, Beyotime, CXCR4 116 NM_009911 China) against GFP to track exogenously delivered MSC β-catenin 155 NM_ 053357 labeled by GFP. A four-channel confocal laser scanning β-actin 103 NM_007393 microscope was used to analyze all the samples. GFP- Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 5 of 16

Fig. 2 MSC cells used in this study. a MSC from SD rat had a fusiform shape and was arranged in bundles or whorls. b MSC established from cells in the primary culture was stained for high-affinity receptors CD90 and CD44 and the low-affinity receptor CD34, CD45, CD11b/c with specific antibodies, and were then analyzed using flow cytometry. c The alkaline phosphatase staining method was used to identify primary cultured osteoblasts

Osteoblast culture medium were obtained from single or combined interventions for Osteoblasts (SD rat strain) were seeded in 6-well plates the follow-up experiment (Fig. 1). for 24 h to 80–90% confluence. Three kinds of interventions were provided: (1) Short-Wave continuous irradi- Wound healing assay ation for 180 min, (2) 200 μmol/l CoCl2-stimulated MSC (SD rat strain) was cultured on 6-well plates to hypoxia condition in fracture, and (3) siRNA inhibition confluency and monolayers and wounded with a sterile of HIF-1α. Eight kinds of osteoblast culture mediums 200-μl pipette tip. The cultures were washed with PBS to remove detached cells and stimulated with 8 kinds of Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 6 of 16

Fig. 3 Radiographs and micro-CT of the femur. a Radiographs of SD rat’s femur. Callus formation was seen in the control group as well as in the SW group (n =4/group). b The normalized radiographic density of the femur in control and different treatment. The higher radiographic density turned out in the SW group at day 14 and day 21 post-operation. After 28 days, no obvious fracture gap was found in each group. c Micro-CT images of the fracture (SD rats). More callus and narrow gaps were seen in the SW group at day14 (n =4/group). d Analysis of ROI bone volume fraction (BV/TV) in the control and SW groups. Arrows in a and c represent fractures. Data represent mean ± SD. Differences were assessed on each day by performing one-way ANOVA. *P < 0.05. Con control, SW Short-Wave treatment osteoblast culture medium fluid 1.5 ml in each well. Pho- Western blotting tographs were collected at 0, 24, and 48 h. Osteoblasts and MSC cell (both are SD rat strain) lysates were prepared, and western blots were performed as previously described [34]. The 30 μg of protein was loaded in Trans-well chamber test each lane for reducing electrophoresis. Primary antibodies The tests were performed in Boyden Chambers (Corn- were used for β-actin (Sigma; A2228) diluted 1:10000, ing, 3422, Lowell, MA). Eight kinds of osteoblast (SD rat HIF-1α (Gene Tex; GTX127309) diluted 1:2000, SDF-1 strain) culture medium were seeded in the bottom (CST; 3740) diluted 1:1000, FAK (Sangon Biotech; chamber. The top chambers filled with MSC (SD rat D160324) diluted 1:3000, phosphor-FAK (Sangon Biotech; strain) starved overnight were inserted. Twenty-four D160324) diluted 1:2000, F-actin (Abcam; Ab205) diluted hours later, inserts were removed and washed. The cells 1:2000, β-catenin (wanleibio; WL0962a) diluted 1:500, and that migrated to the bottom side were accumulated. CXCR4 (Abcam; ab124824) diluted 1:1000. Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 7 of 16

Fig. 4 Tracing MSC homing in nude mice. a Fluorescence assays in vivo. Nude mice of femoral shaft fracture were injected MSC labeled by the GFP (MG) and then randomly divided into two groups, including a Short-Wave treatment group (MG+SW) and a control group (MG) (n = 10/group). MG was examined by an IVIS imaging system 7 days after the operation. b Analysis of total radiant efficiency in the right femur. c Homing of MG to the fracture site, analyzed by histological analysis and immunofluorescence imaging at 28 days after the operation. The rectangular boxes in HE staining sections represent fracture areas, which is the region of interest in immunofluorescence imaging. The MG shown in red accounted for Cy3-labeled antibody. Scale bars 200 μm. d Six high-magnification fields (× 100) were randomly observed, and the number of GFP-positive cells was counted and analyzed. Significantly more MG cells were counted in the femora of animals treated with Short-Wave irradiation. Data represent mean ± SD. Differences were assessed by performing a t test. *P < 0.05. MG injected MSC labeling with the GFP, SW Short-Wave treatment

Statistical analysis The results of flow cytometry demonstrated that over Statistical analysis was performed by SPSS 22.0 for Win- 90.2% and 83.5% of the mononucleated cell colonies iso- dows (SPSS, Chicago, USA). The results are shown as lated from the bone marrow of SD rats were positive for fi- the mean value ± standard deviation. The differences be- broblastic marker CD90 and MSC marker CD44, tween groups were analyzed by t test or analysis of vari- respectively, and negative for hematopoietic lineage ance (ANOVA). Two-tailed P values were computed, markers (CD45 and CD11b/c) and the endotheliocyte and P < 0.05 was considered to be statistically significant. lineage marker CD34 (0.36%, 0.29%, and 0.93%, respectively) (Fig. 2b). Results MSC identification Short-Wave treatment enhancing fracture healing After 3 to 6 passages, MSCs extracted from SD rats were in To examine the role of Short-Wave treatment and fusiform shape, arranged in bundles or whorls (Fig. 2a). MSC during fracture repair, we generated a closed Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 8 of 16

Short-Wave therapy treatment promoting MSC homing in vivo Nude mice were injected with MSC expressing the GFP report gene for observation of the MSC homing to the fracture. IVIS imaging system recorded radiant efficiency in the leg at day 7 post-operation. Higher cell migration was more common in animals receiving Short-Wave irradiation with total radiant efficiency [p/s]/[μW/cm2] compared to those not exposed to wave irradiation (1123.1 ± 116.0 vs 878.2 ± 79.2, P =0.023; Fig. 4a, b). Besides, GFP distribution analysis showed that MSC was not uniformly distrib- uted throughout the body and tended to migrate to organs such as the lung, liver, and tail, which was observed in both groups. Next, we quantified the homing of exogenously de- liveredMSCtothefracturesitebyusingimmuno- fluorescent staining and histological analysis. All the nude mice were sacrificed, and the callus tissues were prepared for immunofluorescence staining at day28aftertheoperation.SignificantlymoreMG cells were counted in the femora of animals treated with Short-Wave irradiation (GFP-positive cells per × 100 field 56.3 ± 26.8 vs 184.3 ± 21.2, P = 0.003; Fig. 4c, d).

Short-Wave therapy changes the gene expression in Fig. 5 Factors’ expression in vivo. a CallusofsacrificedSDratswas collected at days 3, 7, and 14. The expression of HIF-1, SDF-1, FAK, and F- callus actin in callus tissue detected by q RT-PCR (n = 4/group/time point). b The results above indicated that Short-Wave therapy The concentration of SDF-1 in plasma of sacrificed SD rats detected by enhanced MSC migration to the fracture. Low oxy- ELISA at days 3, 7, and 14 (n = 4/group/time point). Data represent gen occurs in the fracture following bony injury [35]. mean ± SD. Differences were assessed among the four groups on each Therefore, the involvement of HIF-1 and its related daybyperformingone-wayANOVA.*P < 0.05. Con control, SW Short-Wave treatment factors (SDF-1, F-actin, and FAK) were investigated after Short-Wave therapy using q RT-PCR. No significant differences in HIF-1 were detected between the two groups at day 3 (P = 0.706) and 14 (P = femoral shaft fracture model. Radiographic examina- 0.602) post-therapy, yet its expression significantly tions showed a normal bone healing process in all increased in the SW group at day 7 compared to the rats. The radiographic analysis demonstrated that the control group (P = 0.002; Fig. 5a). Moreover, com- SW group showed better fracture healing than the pared with the control group, the expression of SDF- control group (Fig. 3a). Besides, the quantitative 1 increased in the SW group on days 7 (P = 0.044) measurement density of the fracture gap showed that and 14 (P = 0.016). In addition, a significant increase SW groups were significantly larger than the control of F-actin was found at day 7 in the SW group (P = group at days 14 (P = 0.033) and 21 (P = 0.026). 0.038), while there was no difference at day 3 (P = Nevertheless, no significant difference was found be- 0.728) and day 14 (P = 0.835). For FAK, it seemed tween days 7 (P = 0.152) and 28 (P = 0.163) (Fig. 3b). that Short-Wave therapy led to increases on days 3 Micro-CT analysis showed that a more significant and 7; nevertheless, there was no statistical differ- amount of bony callus was found in the SW groups than in ence between the two groups at day 3 (P = 0.051), the control group. Three-D reconstructed micro-CT im- day 7 (P = 0.142), and day 14 (P =0.287). ages on day 14 and 28 post-fracture are shown in Fig. 3c. In addition, SDF-1 in plasma was detected by ELISA Comparing with the control group, the bone volume frac- (Fig. 5b). After 7 days of treatment with Short-Wave tion (BV/TV) in the SW group was significantly higher at therapy, SDF-1 was increased by 1.5-folds compared to day 14 (P = 0.045) and day 28 (P = 0.049) (Fig. 3d). the control group (P = 0.044). Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 9 of 16

Fig. 6 Factors’ expression in osteoblasts in vitro. Osteoblasts of SD rats were fed in four kinds of medium. Short-Wave irradiation was provided to the cells in the SW group. a, b The concentration of HIF-1 and SDF-1 in the culture medium of osteoblasts was analyzed by ELISA. c, d The expression of HIF-1 and SDF-1 in the osteoblasts was analyzed by western blot. Data represent mean ± SD. Differences between the control group and the Short-Wave treatment group were assessed by performing a t test. *P < 0.05. Con control, SW Short-Wave treatment

Short-Wave therapy promotes HIF-1 expression in concentration in culture media of osteoblasts was decreased osteoblasts in vitro after treating cells with HIF-1α-siRNA. We evaluated the HIF-1 in the culture medium fluid of osteoblasts under a Short-Wave in vitro by ELIS The migratory effect of HIF-1 on MSC under Short-Wave A. The results showed that the expression of HIF-1 irradiation significantly increased following Short-Wave treat- As shown in Fig. 7, MSC cultured in normoxia with the ment (P = 0.047), especially under CoCl2 stimulated medium fluid of SW-irradiated osteoblasts infected vector hypoxic conditions (P = 0.018). Next, we collected for24h.At48h,itshowedhighermigrationcom- the osteoblasts and measured the expression of HIF- pared to control cells cultured in the medium fluid 1 by western blot. We found a higher expression of without SW irradiation (P < 0.05). The same ten- HIF-1 after Short-Wave irradiation, both under nor- dency was also seen in simulated hypoxic conditions moxia (P = 0.021) and hypoxic condition (P = 0.046) at 24 h (P < 0.05), but not at 48 h. The results of the (Fig. 6a, c). Also, the expression of SDF-1 in the medium im- analysis are seen in Table 2. proved following Short-Wave therapy under normoxia Additionally, to determine the effects of the HIF-1 in (Fig. 6b, d). HIF-1α-siRNA inhabited the HIF-1 expression. Short-Wave treatment on MSC migration, we used The expression of SDF-1 in osteoblasts and its siRNA to inhibit HIF-1 in cultured osteoblasts. Briefly, siRNA reversed the above outcome (Fig. 7, Table 2). Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 10 of 16

Fig. 7 (See legend on next page.) Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 11 of 16

(See figure on previous page.) Fig. 7 Migration in vitro. In wound healing assay, MSC (SD rat strain) was fed with 8 different kinds of osteoblast culture medium. Photographs were collected at 0, 24, and 48 h. Additionally, the chemotactic effect of the culture medium of Short-Wave-irradiated osteoblasts on MSC in vitro analyzed by the trans-well assay. Eight kinds of osteoblast (SD rat strain) culture medium were seeded in the bottom chamber. The top chambers filled with MSC (SD rat strain) starved overnight were inserted. Twenty-four hours later, the cells that migrated to the bottom side were accumulated

Migration-correlation factor expression in MSC fully understood. Previously, it has been reported that Next, we examined whether the medium fluid of osteo- fracture healing is associated with an increase in calcium blasts irradiated by Short-Wave could affect the gene ex- phosphate mineral salt deposition, which occurs 2 to 4 pression of con-cultured MSC. The gene expression of weeks after injury [36, 37]. In this study, we found that CXCR4, β-catenin, FAK, and F-actin in MSC were ana- high-frequency Short-Wave irradiation could promote lyzed using q RT-PCR (Fig. 8a) and western blot (Fig. 8b, the healing process, including the promotion of bony c). As a receptor, CXCR4 was increased under normoxia callus formation and the MSC migration. These healing (P = 0.011) and under hypoxic conditions (P = 0.015). In characteristics were observed 1 to 3 weeks after the addition, it decreased under HIF-1-inhabited medium injury. both under normoxia (P = 0.900) and hypoxia (P = MSC has a vital role in the process of fracture healing 0.046). The Short-Wave-irradiation medium also pro- [38]. The early stage of healing is mainly mediated by vided a positive effect on the expression of β-catenin the biological actions of MSC [38]. Preclinical studies (P = 0.013) and F-actin (P = 0.031) in MSC under hyp- have suggested that transplanted exogenous or endogen- oxia; yet, no statistical difference was observed under ous MSC can migrate to the fracture site after injury normoxia (each P > 0.05). The rise of F-actin and β- and restore the fracture callus volume and biomechan- catenin were restrained since osteoblasts were trans- ical properties of the bone in mice [39]. Injection of planted in HIF-1 siRNA under normoxia and hypoxia MSC by intravenous or intra-arterial infusion is com- (each P > 0.05). Additionally, no statistical difference was monly used to treat bone injury in mice. Yet, systemic observed in FAK between the two groups (each P > administration has low therapeutic efficacy because only 0.05). Nevertheless, phosphorylation levels of FAK were a small percentage of MSC can reach the target tissue much higher in the Short-Wave group under normoxia [40, 41]. Hence, one of the solutions to improve thera- (P = 0.040), which also decreased since MSC cultured in peutic efficacy is to promote MSC migration and the medium of HIF-1 restrained osteoblasts (each P > homing. 0.05). So far, only a few studies have focused on investigating the MSC migration in the electric field, especially in the direct current electric field. Zimolag and Griffin have ob- Discussion served that in an external direct current electric field, The clinic application of high-frequency treatment can MSC directly migrated toward the cathode [42, 43]. accelerate the resolution of hematoma and fracture heal- Moreover, Banks et al. have discovered that cells display ing. Nevertheless, the underlying mechanisms are not a highly elongated phenotype conversion and consistent perpendicular alignment to the electric field vector ac- Table 2 Migration test in vitro counting for the effects of electric field strength [44]. Wound region (%WR)a Number Furthermore, Liu and Zhao found that the applied elec- of 24 h 48 h tromagnetic field might be useful to control or enhance migrated cells 24 h the migration of MSC during bone healing [45, 46]. Vector 31.4 ± 1.1 19.8 ± 0.8 319 ± 13 Nevertheless, as a kind of high-frequency electrotherapy, areas of exposure could not be polarized by Short-Wave Vector+SW 27.7 ± 1.5b 8.84 ± 1.3b 420 ± 15b therapy, the migration of MSC might not be associated Hif-1-SiRNA 44.9 ± 1.7 38.7 ± 3.7 221 ± 11 with field stress. Short-Wave therapy is generally used Hif-1-SiRNA+SW 46.1 ± 1.4 40.0 ± 1.5 229 ± 12 for thermal effects that provoke or enhance cellular ac-

CoCl2+Vector 45.6 ± 0.9 32.2 ± 3.0 244 ± 10 tivity resulting from energy-absorbing in oscillating elec- c c CoCl2+Vector+SW 35.4 ± 2.2 33.8 ± 3.4 374 ± 17 trical fields [47]. Previous studies have shown that thermal stress can modulate some molecules affected by CoCl2+Hif-1-SiRNA 45.9 ± 1.2 42.6 ± 1.7 221 ± 11 hypoxia [48, 49]. For example, HIF-1α, a transcription CoCl +Hif-1-siRNA+SW 43.7 ± 1.6 38.8 ± 1.8 207 ± 13 2 factor that is positively correlated with the acute a%WR = (the size of the wound region/total size of image) × 100. bVector VS c temperature changes in organs, such as the brain, liver, Vector+SW at the same time point P < 0.05. CoCl2+Vector VS CoCl2+Vector+SW at the same time point P < 0.05 kidney, and gonad tissues, can regulate the cellular Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 12 of 16

Fig. 8 Factors’ expression in MSC. a MSC (SD rat strain) was fed with different kinds of osteoblast culture medium. The gene expression of CXCR4, β-catenin, FAK, and F-actin in MSC detected by q RT-PCR. b The presence of the factors as the protein of MSC was detected by western blot. c Quantification of protein bands from western blot films. The quantification will reflect the relative amounts as a ratio of each protein band relative to the lane’s loading control β-actin. The data of western blot represent mean ± SD. Differences between the control group and SW group were assessed by performing a t test. *P < 0.05. Con control, SW Short-Wave treatment response to hypoxia stress [50] that is significantly in- and survival [52, 53]. In this study, it was found that ex- creased in osteoblasts [51]. In the current study, we did ogenous MSC homing increased in fractures exposed to not focus on the direct effect of Short-Wave on the mi- Short-Wave irradiation in vivo. Besides, we discovered gration of MSC in vitro. We found an increased expres- that the MSC migration was improved by the cultural sion of HIF-1 in callus and osteoblasts exposed to Short- medium of osteoblast exposed to Short-Wave therapy ir- Wave therapy, which could be the chemokines for MSC radiation in vitro, which could be further enhanced in to fracture area. Consequently, we assume that the regu- stimulated hypoxia in fracture and restrained by inhibit- lation of the hypoxia pathway in callus affects MSC ing HIF-1α expression of culture osteoblasts. Therefore, homing to promote tissue regeneration by Short-Wave. we believe that HIF-1 is a key factor in the healing High expression of HIF-1a and BMP-2 promote the mi- process activated by Short-Wave treatment. However, gration of MSC to the bone defect area [3]. Therefore, in the effect of Short-Wave treatment on fracture healing several tissue engineering strategies, HIF stabilized bio- of the HIF-1a inhibitor was not observed in vivo, which logical materials are used to improve MSC migration is a limitation of the current study. A research of HIF-1a Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 13 of 16

Fig. 9 Schematic diagram of Short-Waves promoting fracture healing by chasing HIF-1 and promoting MSC migration. SW Short-Wave treatment, HIF-1 hypoxia-inducible factor 1, SDF-1 stromal cell-derived factor 1, CXCR4 C-X-C motif receptor 4, PI3K phosphatidylinositol 3-kinase, Erk extracellular regulated protein kinases, FAK focal adhesion kinase, MSCs mesenchymal stem cells conditional knockout rats of fracture with Short-Wave injured tissues can be prevented if SDF-1 in ischemic tis- therapy will be conceived in the future. sue or CXCR4 on circulating cells were blockaded [54, The homing of CXCR4-positive progenitor cells in cir- 57]. In the bone marrow, discrete regions of the anoxic culation is upregulated since the increase of HIF-1 in- chamber have increased SDF-1 expression and progeni- duces SDF-1 expression. As a cell growth-stimulating tor cell tropism [58]. Over the last 10 years, numerous factor, SDF-1 belongs to the CXC subfamily of chemo- studies have confirmed that SDF-1/CXCR4 has a pivotal kines [54, 55]. SDF-1 can activate CXCR4, a G protein- role in the biologic and physiologic functions of MSC coupled receptor [56]. Progenitor cell recruitment to [59]. In this study, we found increased expression of Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 14 of 16

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Stem Cell Res Ther. 2017;8(1):208. https://doi.org/ Author details 10.1186/s13287-017-0659-2. 1Department of Rehabilitation, Affiliated Zhongshan Hospital of Dalian 19. Ishii M, Takahashi M, Murakami J, Yanagisawa T, Nishimura M. Vascular University, Dalian 116001, China. 2Department of Anatomy, Medical College endothelial growth factor-C promotes human mesenchymal stem cell of Dalian University, Dalian, China. 3Department of Rehabilitation, The migration via an ERK-and FAK-dependent mechanism. Mol Cell Biochem. people’s Hospital of Longhua District, Shenzhen, China. 2019;455(1–2):185–93. https://doi.org/10.1007/s11010-018-3481-y. Ye et al. Stem Cell Research & Therapy (2020) 11:382 Page 15 of 16

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